CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants.

BACKGROUND: Genomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation. RESULTS: We implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologiesCleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage. CONCLUSIONS: We demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches.

Homozygosity Mapping Reveals Population History and Trait Architecture in Self-Incompatible Pear (Pyrus spp.).

Runs of homozygosity (ROH) have been widely used to study population history and trait architecture in humans and livestock species, but their application in self-incompatible plants has not been reported. The distributions of ROH in 199 accessions representing Asian pears (45), European pears (109), and interspecific hybrids (45) were investigated using genotyping-by-sequencing in this study. Fruit phenotypes including fruit weight, firmness, Brix, titratable acidity, and flavor volatiles were measured for genotype-phenotype analyses. The average number of ROH and the average total genomic length of ROH were 6 and 11 Mb, respectively, in Asian accessions, and 13 and 30 Mb, respectively, in European accessions. Significant associations between genomic inbreeding coefficients (FROH) and phenotypes were observed for 23 out of 32 traits analyzed. An overlap between ROH islands and significant markers from genome-wide association analyses was observed. Previously published quantitative trait loci for fruit traits and disease resistances also overlapped with some of the ROH islands. A prominent ROH island at the bottom of linkage group 17 overlapped with a recombination-supressed genomic region harboring the self-incompatibility locus. The observed ROH patterns suggested that systematic breeding of European pears would have started earlier than of Asian pears. Our research suggest that FROH would serve as a novel tool for managing inbreeding in gene-banks of self-incompatible plant species. ROH mapping provides a complementary strategy to unravel the genetic architecture of complex traits, and to evaluate differential selection in outbred plants. This seminal work would provide foundation for the ROH research in self-incompatible plants.

Marker-trait associations and genomic predictions of interspecific pear (Pyrus) fruit characteristics.

Interspecific pear (Pyrus spp.) hybrid populations are often used to develop novel cultivars. Pear cultivar breeding is a lengthy process because of long juvenility and the subsequent time required for reliable fruit phenotyping. Molecular techniques such as genome-wide association (GWA) and genomic selection (GS) provide an opportunity to fast-forward the development of high-value cultivars. We evaluated the genetic architecture of 10 pear fruit phenotypes (including sensory traits) and the potential of GS using genotyping-by-sequencing of 550 hybrid seedlings from nine interrelated full-sib families. Results from GWA suggested a complex polygenic nature of all 10 traits as the maximum variance explained by each marker was less than 4% of the phenotypic variance. The effect-size of SNPs for each trait suggested many genes of small effect and few of moderate effect. Some genomic regions associated with pear sensory traits were similar to those reported for apple - possibly a result of high synteny between the apple and pear genomes. The average (across nine families) GS accuracy varied from 0.32 (for crispness) to 0.62 (for sweetness), with an across-trait average of 0.42. Further efforts are needed to develop larger genotype-phenotype datasets in order to predict fruit phenotypes of untested seedlings with sufficient efficiency.

Validation of SNP markers for fruit quality and disease resistance loci in apple (Malus × domestica Borkh.) using the OpenArray® platform.

Genome mapping has promised much to tree fruit breeding during the last 10 years. Nevertheless, one of the greatest challenges remaining to tree fruit geneticists is the translation of trait loci and whole genome sequences into diagnostic genetic markers that are efficient and cost-effective for use by breeders, who must select genetically optimal parents and subsequently select genetically superior individuals among their progeny. To take this translational step, we designed the apple International RosBREED SNP Consortium OpenArray v1.0 (IRSCOA v1.0) assay using a set of 128 apple single nucleotide polymorphisms (SNPs) linked to fruit quality and pest and disease resistance trait loci. The Thermo Fisher Scientific OpenArray® technology enables multiplexed screening of SNP markers using a real-time PCR instrument with fluorescent probe-based Taqman® assays. We validated the apple IRSCOA v1.0 multi-trait assay by screening 240 phenotyped individuals from the Plant & Food Research apple cultivar breeding programme. This set of individuals comprised commercial and heritage cultivars, elite selections, and families segregating for traits of importance to breeders. In total, 33 SNP markers of the IRSCOA v1.0 were validated for use in marker-assisted selection (MAS) for the scab resistances Rvi2/Vh2, Rvi4/Vh4, Rvi6/Vf, fire blight resistance MR5/RLP1, powdery mildew resistance Pl2, fruit firmness, skin colour, flavour intensity, and acidity. The availability of this set of validated trait-associated SNP markers, which can be used individually on multiple genotyping platforms available to various apple breeding programmes or re-designed using the flanking sequences, represents a large translational genetics step from genomics to crop improvement of apple.

Fine-mapping and validation of the genomic region underpinning pear red skin colour.

Red skin colour is an important target trait in various pear breeding programmes. In this study, the genetic control of red skin colour was investigated in an interspecific population derived using the descendants of the red sport European pear cultivar 'Max Red Bartlett' (MRB) and the red-blushed Chinese pear cultivar 'Huobali'. Approximately 550 seedlings from nine families were phenotyped for red skin over-colour coverage (Ocolcov) and the intensity of red over-colour (Ocolint) on a 0-9 scale, and genotyped using genotyping-by-sequencing. Genome-wide association analyses were conducted using 7500 high-quality single nucleotide polymorphisms (SNPs). Genomic regions on linkage groups (LG) 4 and 5 were found to be associated, and the best SNP (S578_25116) on LG4 accounted for ~15% of phenotypic variation in Ocolcov and Ocolint. The association of S578_25116 with Ocolcov and Ocolint was successfully validated in a sample of ~200 European and Asian pear accessions. The association with red skin at locus S578_25116 was not present in Asian pear accessions, suggesting its close proximity to the MRB's Cardinal gene. Several putative candidate genes, including MYB transcription factors (PCP027962 and PCP027967), were identified in the quantitative trait locus region on LG4 and await functional validation.

Putting the Community back into Community Health Needs Assessments: Maximizing Partnerships Via Community-Based Participatory Research.

THE PROBLEM: The community health needs assessment (CHNA) mandate of the Patient Protection and Affordable Care Act (ACA) has the potential to make significant and sustainable change in the health of communities. However, to date many hospital-led assessments have used traditional, top-down data collection approaches that overemphasize individualized community member deficits and underutilize collaboration across sectors. PURPOSE: The purpose of this paper is to present the principles of community-based participatory research (CBPR) as a framework for conducting CHNAs in a way that mitigates the potential for harm, waste, and misrepresentation of community assets and needs that characterizes many existing CHNA processes, illustrating the power of applying CBPR partnerships to this process. KEY POINTS: CBPR is a framework to engage community members directly in research design, the collection and analysis of data, and the creation of action plans that address research findings. Key principles include collaborative involvement, establishment of empowering processes, and long-term commitment. A case example of an innovative community partnership demonstrates the power and challenges of taking a CBPR approach to the CHNA process. CONCLUSIONS: CBPR has incredible potential to be incorporated into ACA-mandated hospital CHNAs, leading to increased impact and shared power with community members.

Genotyping-by-sequencing of pear (Pyrus spp.) accessions unravels novel patterns of genetic diversity and selection footprints.

Understanding of genetic diversity and marker-trait relationships in pears (Pyrus spp.) forms an important part of gene conservation and cultivar breeding. Accessions of Asian and European pear species, and interspecific hybrids were planted in a common garden experiment. Genotyping-by-sequencing (GBS) was used to genotype 214 accessions, which were also phenotyped for fruit quality traits. A combination of selection scans and association analyses were used to identify signatures of selection. Patterns of genetic diversity, population structure and introgression were also investigated. About 15 000 high-quality SNP markers were identified from the GBS data, of which 25% and 11% harboured private alleles for European and Asian species, respectively. Bayesian clustering analysis suggested negligible gene flow, resulting in highly significant population differentiation (Fst=0.45) between Asian and European pears. Interspecific hybrids displayed an average of 55% and 45% introgression from their Asian and European ancestors, respectively. Phenotypic (firmness, acidity, shape and so on) variation between accessions was significantly associated with genetic differentiation. Allele frequencies at large-effect SNP loci were significantly different between genetic groups, suggesting footprints of directional selection. Selection scan analyses identified over 20 outlier SNP loci with substantial statistical support, likely to be subject to directional selection or closely linked to loci under selection.

Exploring civic engagement at an urban commuter campus: pathways and barriers.

Universities have traditionally had a vested interest in the civic preparation of students. In order to understand the unique situation of this population of students in a university serving non-traditional community students, qualitative data was used to identify pathways and barriers associated with their civic engagement. Results suggest themes associated with time, knowledge, and access.

Sexual attitudes and behaviors among adolescents.

The purpose of this article is to examine the self-reported sexual attitudes and behaviors of adolescents living in the Midwest. A survey was administered to 463 African American youths ages 11-19. The results indicate that intent to have sexual intercourse, reported likelihood toward having sexual intercourse, and whether participants felt that they would have sex in the next 3 months were predictors of whether they had sexual intercourse at the 3-month follow-up. The results also revealed that the opinions of referent others did not predict sexual intercourse at 3 months. Negative attitudes toward condoms did predict condom use at 3 months and the opinions of referent others also predicted condom use at 3 months. Limitations as well as future research are also discussed.

Substance use among African American adolescents in the Midwest.

The purpose of this article is to examine the attitudes and substance use behaviors of African American adolescents living in the Midwest. A baseline survey was administered to 463 African American teens between the ages of 11-19. The article examines the relationship between attitudes toward drugs and drug-using behavior in this African American sample. Drug use will be compared to national drug use norms established by the Youth Risk Behavior Surveillance Survey. Overall participants had fairly negative attitudes toward drugs. Sixty percent of the sample reported that they were committed to a drug-free life, 74% had made a decision to stay away from marijuana, 79% reported making a decision not to smoke cigarettes, and 71% reported they would not get drunk in the next year. Females were more likely to stay away from marijuana than males. In this current study there is cause for alarm; participants reported higher percentages of ever smoking cigarettes and marijuana than the Youth Risk Behavior Surveillance Survey. This study shows there is a need to provide substance abuse prevention programs for African American adolescents. Limitations and future directions are also discussed.

The power of aspirations and expectations: the connection between educational goals and risk behaviors among African American adolescents.

Numerous studies and interventions have been designed to either reduce the prevalence of risky behaviors (substance use, sexual activity, etc.) or increase the level of educational activity for adolescents. Research suggests that these two constructs may be related yet little is known about this relationship among African American youth. Archival data from the Risk Reduction Project was used to explore the connection between the protective factors of educational aspirations/expectations and recent participation in risky behavior. Results suggest that recent use of marijuana, alcohol to the point of intoxication, and sexual activity predicted lower aspirations and expectations among African American youth. From a positive youth development perspective, this study suggests the development of holistic interventions that build educational assets in youth, in addition to working to minimize risk behaviors. Limitations and suggestions for future research are also discussed.

Assessing the health behaviors of African American emerging adults.

The purpose of this article was to assess the overall health behaviors of African American emerging adults who live in a Midwestern city. Two hundred participants, ages 18-25, completed a 129-item survey. Results indicate that African American emerging adults are engaged in relatively healthy behaviors (i.e., physical activity, low substance use). The results also showed that within the emerging adult developmental period, there are differences in behavior for those who are in the younger (18-20) versus those that are older (21-25) in the developmental period. Limitations, future research, and implications for practice will also be discussed.

Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1.

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A-RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.